Journal of Hazardous Materials 260 (2013) 375– 382
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Journal of Hazardous Materials
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iochar properties regarding to contaminants content andcotoxicological assessment
atryk Oleszczuk ∗, Izabela Josko, Marcin Kusmierzepartment of Environmental Chemistry, Faculty of Chemistry, 3 Maria Curie-Skłodowska Square, 20-031 Lublin, Poland
i g h l i g h t s
Biochars from commercial manufac-turer were analysed.Heavy metals and PAHs were ana-lysed in context of ecotoxicologicalproperties.High concentration of PAHs wasobserved for some biochars.Almost all tested biochars were toxicin bioassays applied.
g r a p h i c a l a b s t r a c t
a r t i c l e i n f o
rticle history:eceived 25 February 2013eceived in revised form 1 May 2013ccepted 24 May 2013vailable online 30 May 2013
a b s t r a c t
The objective of the study was the determination of the content of contaminants and toxicity of fourdifferent biochars. The properties of the biochars, content of trace metals and polycyclic aromatic hydro-carbons (16 PAHs) were determined. Toxicological estimation of the biochars was performed on the basisof a battery of biotests with plants (Lepidium sativum), bacteria (Vibrio fischeri and 11 different strains from
MARA), alga (Selenastrum capricornutum), protozoa (Tetrahymena thermophila) and crustaceans (Daphniamagna). The content of trace metals depended on the biochar and was comparable to uncontaminatedsoils. PAHs sum varied from 1124 to 28,339 �g/kg. The toxicity of the biochars depended both on theirkind and on the test applied. The most sensitive organism was D. magna. Relatively the least sensitiveto extracts from the biochars proved to be S. capricornutum and T. thermophila. A significant correlationbetween the content of PAHs and toxicity was noted only in the case of D. magna.
The application of biochar in soils is becoming an increasinglyommon treatment. Biochar is an interesting solution as on the oneand it improves the properties of soils [1,2], and on the other,hich is extremely important, it may constitute a tool in the com-
at against the global worming through the sequestration of CO2
3]. Recent studies showed, similarly to activated carbon (AC), thatiochar may be use for the immobilisation of organic and inorganicontaminants in various environmental matrices, i.e. sediments,
soils and sewage sludges [4–6]. This results from the fact thatbiochar is characterised by high affinity to organic and inorganiccontaminants [7]. Due to binding of contaminants by biochar, themobility and bioavailablity of contaminants decrease, and thus alsotheir potential toxicity to various groups of organisms.
In spite of the positive effect that biochar may have on the soil, inthe literature attention is paid more and more frequently on biocharcontamination with polycyclic aromatic hydrocarbons (PAHs) andtrace metals [8–11]. Depending on the kind of biochar, the contentof PAHs may vary and exceed considerably the threshold values
relating to biosolids [12]. It is assumed that both the concentra-tion and the composition of individual PAHs depend to a notableextent on temperature and on the kind of biomass used to producebiochar [10,13]. The mechanism of the formation of PAHs in the
rocess of pyrolysis has been studied in detail [10,14,15]. The PAHsormed in that process are then adsorbed on the biochar. Studiesonducted by Ledesma et al. [14] and Masek et al. [15] suggestedhat PAHs are formed mainly at temperatures higher than 700 ◦C.he latest research shows, however, that PAHs can also be formed atower temperatures [10]. Next to the temperature another impor-ant parameter affecting the content of PAHs can be the kind of
aterial used for biochar production [10,13]. The presence of PAHsn biochars puts, therefore, a question mark over the possibilityf their uncontrolled utilisation in soil fertilisation. Another fac-or that may impose a considerable limitation on the utilisation ofiochars is their content of trace metals. Studies concerned withhe problem of trace metals are not as numerous as those relat-ng to PAHs, but they also indicate that those compounds maye present in biochars [8,16], which creates a potential threat tohe environment. The content of trace metals largely depends onheir concentration in the initial materials used for the biocharsroduction [16]. Both from a regulatory and environmental per-pective, using a soil amendment material that contains PAHs andrace metals that pose a threat to the soil is unacceptable [11].
Studies conducted so far concerning biosolids show that chem-cal analysis are not a sufficient tool for the estimation of the riskelated to the utilisation of these materials as a soil fertiliser [17].iological tests may prove to be useful for that purpose. Biotestshould not be treated as an alternative to chemical analyses, butupplementation of studies with biological tests may expand thenowledge on the potential risks. In addition, the application of bio-ogical tests permits the study of the possible interactions amongarious contaminants that provide ultimate evidence on the exist-nce or absence of toxic effect on organisms.
The objective of the study presented herein was the estima-ion of the content of contaminants that may potentially occurn biochars, i.e. trace metals (Cd, Cu, Cr, Ni, Pb, Zn) and PAHs.he results obtained were related with ecotoxicological estima-ion covering a broad group of organisms (bacteria, plants, algae,nvertebrates). The information acquired will permit the character-sation, as exhaustive as possible, of biochars with regard to theirotential utilisation for soil amendment.
. Materials and methods
.1. Biochar characteristic
Four different biochars (Table 1) were used in the present study.ll the biochars investigated here were obtained from commercialanufacturer and were produced by pyrolysis where the feed-
tock is thermochemically decomposed at a temperature rangerom 350 ◦C (start of combustion) to 650 ◦C (max. combustion tem-
erature) in an oxygen-poor atmosphere (1–2% O2). Biochars BC-M,C-O and BC-W were produced of elephant grass (miscanthus),oconut shell and wicker, respectively and were provided by com-ercial manufacturer Fluid S.A. (Sedziszów, Poland). Biochar BC-2
able 1he physico-chemical properties and elemental composition of investigated biochars.
Biochars pH CEC Available forms of
P2O5 K2O Mg
BC-2 9.9a 530a 540a 2824a 163a
BC-W 9.1a 143b 122b 772b 32b
BC-O 8.0b 148b 106a 1456c 156a
BC-M 6.8c 144b 132b 1556c 39b
H in KCl. CEC – the cation exchange capacity (mmol/kg); P2O5, K2O and Mg – available ff carbon, hydrogen and nitrogen; H/C – ratio of hydrogen to carbon; SBET – specific surfaetween biochars.
s Materials 260 (2013) 375– 382
was produced of wheat straw and was provided by Mostostal Sp. zo.o. (Wrocław, Poland).
The chemical properties of biochars were determined bystandard methods. The pH was measured potentiometrically in 1 MKCl after 24 h in the liquid/soil ratio of 10. The cation exchangecapacity (CEC) and available potassium, phosphorus and magne-sium were determined according to procedures for soil analysis[18]. The amounts of carbon, hydrogen and nitrogen were deter-mined using CHN equipment (Perkin–Elmer 2400). To analysethe textural characteristics of materials, low-temperature (77.4 K)nitrogen adsorption–desorption isotherms were recorded using aMicromeritics ASAP 2405 N adsorption analyser. The specific sur-face area SBET was calculated according to the standard BET method[19].
2.2. Contaminants content analysis
Pseudototal metal concentration was extracted in the PROLABOmicrowave oven (Microdigest 3.6, France), using a wet method in amixture of nitric acid (8 ml) and perchloric acid (8 ml) at the ratio of1:1. A 30% of hydrogen peroxide solution was added before acids. Ananalysis for the content of Cd, Cr, Cu, Fe, Ni, Pb, and Zn was carriedout using emission spectrometry on ICP-OES (Leeman Labs, PS 950).Evaluation of the accuracy and precision of analytical proceduresused reference materials (Heavy Clay Soil, RTH 953, Promochem).
Polycyclic aromatic hydrocarbons were analysed according tothe method developed by Hilber et al. [9]. Dry biochar sampleswere extracted using accelerated solvent extractor (ASE 200) fromDionex GmbH (Idstein, Germany). A qualitative and quantitativeanalysis of PAHs was carried out on a liquid chromatograph withDAD and Fluorescence detectors (Waters, USA). The proceduralblank was determined by going through the same extraction andcleanup procedures for each series of samples. None of the ana-lytical blanks were found to have detectable contamination of themonitoring PAHs and thus the results were not blank corrected.
2.3. Bioassays
Biochars toxicity was assessed with the commercial toxicitybioassay – Phytotoxkit FTM Test [20]. The phytotoxkit microbiotestmeasures the decrease (or the absence) of seed germination andof the growth of the young roots after 3 days of exposure of seedsof selected higher plants to contaminated matrix in comparison tothe controls in a reference soil. The analyses and the length mea-surements were performed using the Image Tool 3.0 for Windows(UTHSCSA, San Antonio, USA). The bioassays were performed inthree replicates. Biochar was added to OECD soil at three doses 1%,5% and 10%.
To evaluate the effect of biochars on alga, bacteria, protozoa
and crustaceans extracts from biochars were tested. Extracts wereobtained according to the EN 12457-2 protocol [21]. The sampleswere mixed with de-ionised water in a single-stage batch test per-formed at a liquid-to-solid (L/S) ratio of 1 l/100 g. The glass bottles
orms of phosphorous, potassium and magnesium (mg/kg); CHN – contribution (%)ce area (m2/g). Different letters means statistically significant differences (P ≥ 0.05)
ardous Materials 260 (2013) 375– 382 377
wfi
AFSUtM
baunlo“w
aanTbtMuD9setuTd
natFwss
pft
nitm
tp(m
2
(mun
Table 2Heavy metals content (mg/kg) in investigated biochars.
ere shaken in a roller-rotating device at 10 rpm. The extracts wereltered by filter with a porosity of 0.45 �m.
A battery of 5 bioassays was used for study: Microtox, Microbialssay for toxic Risk Assessment (MARA), Algaltoxkit FTM, ProtoxkitTM and Daphtoxkit FTM. Microtox reagents were purchased fromDI (Delaware, USA), MARA reagents from NCIMB Ltd. (Aberdeen,K) while microbiotests (Algaltoxkit FTM, Protoxkit FTM and Daph-
oxkit FTM) with all reagents and equipment were purchased fromicroBioTests (Creasel, Deinze, Belgium).The Microtox® Toxicity Test was used to evaluate the inhi-
ition of the luminescence in the marine bacteria Vibrio fischericcording to the test protocol [22]. The tests were carried outsing a Microtox M500 analyser. The light output of the lumi-escent bacteria from biochars extract was compared with the
ight output of a blank control sample. Luminescence inhibitionf extract was assessed for 15 min of exposure carrying out the81.9% Basic test protocol” (screening test, MicrotoxOmni softwareas used).
Microbial Assay for Risk Assessment (MARA) is a multi-speciesssay which allows measurement of toxic effects of chemicalsnd environmental samples. The test uses a selection of taxo-omically diverse microbial species lyophilised in a microplate.en prokaryotic species and a eukaryote (yeast) constitute theiological indicators of toxicity assessment. In the present work,he microbial species used consisted of ten bacterial species: 1.icrobacterium sp., 2. Brevundimonas diminuta, 3. Citrobacter fre-
ndii, 4. Comamonas testosteroni, 5. Enterococcus casseliflavus, 6.elftia acidovorans, 7. Kurthia gibsonii, 8. Staphylococcus warnerii,. Pseudomonas aurantiaca, 10. Serratia rubidaea, and one yeastpecies, 11. Pichia anomalia [23]. The growth of the organismsxposed to the test sample is determined with the reduction ofetrazolium red (TZR). A scanned image of the microplate obtainedsing a flatbed scanner is analysed using purpose-built software.he MARA test was performed according to the standard protocolescribed by Wadhia et al. [23].
Algaltoxkit FTM uses the green microalgae Selenastrum capricor-utum, de-immobilised from alginate beads, for determination oflgal growth inhibition after 72 h. Tests were performed accordingo the standard operational procedure manual of the AlgaltoxkitTM [24], which follows OECD Guideline 201 [25]. The algal biomassas evaluated by measuring the optical density of algal suspen-
ions at 670 nm after 72 h of exposure to extracts or contrololutions.
Protoxkit FTM test measures growth inhibition of the ciliaterotozoan Tetrahymena thermophila after 24 h. The tests were per-ormed according to the standard operational procedure manual ofhe Protoxkit FTM [26].
Daphtoxkit FTM acute test with Daphnia magna makes use ofeonates hatched from dormant eggs (ephippia) to determine the
nhibition of motility after 24 h. Tests were performed according tohe standard operational procedure manual of the Daphtoxkit FTM
agna [27], which follows OECD Guideline 202 [28].In order to check the correct performance of the tests and
he sensitivity of the test organisms, a quality control test waserformed with the reference chemical potassium dichromateK2Cr2O7). The whole procedure was carried out according to user’s
anual. The quality control test was successful.
.4. Data analysis
Toxicity data were expressed as the percentage of toxic effect
PE) compared to the control. The differences between each treat-
ent and the control as well as between treatments were evaluatedsing a one-way analysis of variance (ANOVA) followed by Dun-ett’s post hoc test.
nd – not detected. Different letters means statistically significant differences(P ≥ 0.05) between biochars.
3. Results
3.1. Biochar properties
Table 1 presents the properties of the biochars studied. Almostall biochars showed an alkaline character (pH 8.0–9.9). The excep-tion was the biochar derived from miscanthus (BC-M), for of whichpH was at the level of 6.8. The highest values of CEC were notedfor BC-2 (530 mmol/kg) which was obtained from another manu-facturer than three other biochars. For these biochars the valuesof CEC were several-fold lower than for BC-2 and ranged from 143to 148 mmol/kg. The highest levels of available forms of P2O5, K2Oand Mg were also noted, as in the case of CEC for biochar BC-2.However, significant differences between P2O5, K2O and Mg wereobserved among the particular biochars from the same manufac-turer (BC-W, BC-M and BC-O). Irrespective of the kind of biochar,the highest content was noted for K2O (772–28241 mg/kg). OnlyBC-O was characterised by a higher level of Mg than of P2O5. Inthe remaining cases, the content of P2O5 was significantly higherthan that of Mg. The elemental analysis (CHN) indicated relativelysmall differences among the biochars. The highest level of carbonin the samples tested was characteristic of BC-W and BC-M. In thecase of those biochars also the highest level of H was observed.The content of nitrogen was the highest in BC-O and BC-2. Molarratios of elements (H/C ratio) were determined to estimate the aro-maticity and carbonisation of biochars. All tested biochars werecharacterised by a very low H/C ratio confirming a high level of car-bonisation and aromatisation of these materials [29] (Table 1). Thedegree of carbonisation of the biochars studied was as follows: BC-W < BC-M < BC-O < BC-2 and did not differ significantly among theparticular biochars. Whereas, the biochars differed notably fromone another in terms of their surface area (SBET). The biochar withthe highest specific surface area was BC-2. Values smaller by morethan a half were noted for BC-W, while BC-O and BC-M had thelowest values of SBET.
3.2. Trace metals content
The content of trace metals was distinctly related with the kindof biochar. Only in the case of Pb the occurrence of that elementwas found to be at a similar level with no significant differencesamong the particular biochars (from 20.6 to 23.7 mg/kg). In thecase of the remaining metals notable differences were observed.In ascending concentration these were: Cd (0.04–0.87 mg/kg), Cu(0.00–3.81 mg/kg), Ni (0.00–9.95 mg/kg), Cr (0.00–18 mg/kg) andZn (30.2–102.0 mg/kg) (Table 2). The biochar derived from mis-canthus (BC-M) was characterised by the highest levels of Cd, Ni, Znand Cr. The content of Cd in BC-M was several-fold higher compar-ing to the remaining biochars. The biochar derived from coconut(BC-O) was characterised by the highest content of Cu. The lowest
frequency of occurrence in the biochars studied was noted for Ni.The presence of Ni was observed only in biochar BC-M.
Estimating the content of trace metals in relation to the Polishand EU norms concerning sewage sludge (Table S1), in no case their
378 P. Oleszczuk et al. / Journal of Hazardous Materials 260 (2013) 375– 382
PAHs
Na Ace Ac Fl Phen Ant Fl uo Pyr BaA Ch BbF BkF BaP
Con
cent
ratio
n [n
g/g]
0
100
200
300
400
500
600
700
800BC-W
PAHs
Na Ace Ac Fl Phen Ant Fl uo Pyr BaA Ch BbF BkF BaP0
100
200
300
400
500
600
700BC-O
Na Ace Ac Fl Phen Ant Fl uo Pyr BaA Ch BbF BkF BaP
Con
cent
ratio
n [n
g/g]
0
2000
4000
6000
8000
10000
12000
14000BC-M
Na Ace Ac Fl Phen Ant Fl uo Pyr BaA Ch BbF BkF BaP0
500
1000
1500
2000
2500
3000BC-2
PAHs su m = 1124.2 ng/g PAHs su m = 1223.5 ng/g
PAHs su m = 28339.1 ng/g PAHs su m = 7155.7 ng/g
perim
esau
3
TmwAswif1to3(rIe
3
dttWwidsc
significant increase in their toxicity that did not differ significantlybetween the materials and amounted to 48% and 33%, respectively.The highest doses of BC-M and BC-2 (10%) caused further increase
PAHs
Fig. 1. Polycyclic aromatic hydrocarbons content in biochars used in the ex
xceeding was noted. The assayed levels of the metals were con-iderably lower than the permissible norms, which may suggestn absence of threat on their part under conditions of biocharstilisation as a soil fertiliser.
.3. Polycyclic aromatic hydrocarbons content
Fig. 1 presents the content of PAHs in the biochars studied.he level of the sum of PAHs clearly depended on the kind ofaterial tested. The highest concentration of the sum of PAHsas noted for biochar from miscanthus (BC-M) (28 339 �g/kg).
relatively high level of PAHs was also found in the case of thetraw-derived biochar (BC-2). In this case the content of PAHsas at the level of 7156 �g/kg. The content of the sum of PAHs
n the two remaining biochars, i.e. BC-W and BC-O, did not dif-er significantly from each other and amounted to 1124 �g/kg and224 �g/kg, respectively. The dominant group of PAHs in terms ofheir content were 3-ring compounds which constituted dependingn the biochars from 64.6% to 82.6% of total PAHs content. Within-ring PAHs the notably dominant compound was phenanthrene28.7–53.1%). Other compounds with a high contribution were fluo-ene (11.3–26.8%), anthracene (5.2–13.4%) and pyrene (5.0–13.8%).n none of the biochars studied was presence detected of the heavi-st PAHs, i.e. DahA, BghiP and Ind.
.4. Ecotoxicological properties
The ecotoxicological estimation of the biochars studiedepended clearly both on the test applied and on kind of biocharested (Figs. 2–5). In none of the cases was any significant effect ofhe materials studied on seed germination found (data not shown).
hereas, significant differences between the particular biocharsere observed for root growth inhibition (Fig. 2). The lowest tox-
city towards Lepidium sativum was characteristic of the biocharerived from wicker (BC-W). Irrespective of the dose applied ittimulated root growth at the level of 12–41% with relation to theontrol OECD soil. However, no significant relationship was noted
PAHs
ent. Error bars represents standard deviation error (SD, n = 3 extractions).
between the dose and the effect observed. A stimulating effect wasalso noted for the biochar derived from coconut (BC-O). As it wasobserved for BC-W, also in this case no significant relation wasfound between the dose and the effect. The highest dose of BC-O, however, caused a slight inhibition of root growth, at the levelof about 4%. Relatively the most unfavourable effect of the biocharsunder study was noted for the biochar derived from miscanthus(BC-M) and from wheat straw (BC-2). Only the lowest doses (1%)of those materials did not cause any significant inhibition of rootgrowth. Increase of the doses of both BC-M and BC-2 to 5% caused a
Fig. 2. Effect of biochars on root growth inhibition of L. sativum depending on theirdose. Values are given as averages with standard deviations (n = 3).
P. Oleszczuk et al. / Journal of Hazardou
Biochars
BC-W BC-O BC-M BC-2
Lum
ines
cenc
e in
hibi
tion
[%]
0
20
40
60
80
100
a
b
c
d
FV
ow
bwp(b
relation to S. capricornutum (Algaltoxkit FTM) and Tetrahymena ther-
ig. 3. Inhibition of V. fischeri luminescence after exposure to biochars’ leachates.alues are given as averages with standard deviations (n = 3).
f toxicity which for BC-M attained nearly 92%. The toxicity of BC-2as not as high as that of BC-M and was at the level of 58% (Fig. 2).
As in the case of the Phytotoxkit FTM test, also in the case ofacteria significant differences between the biochars under studyere observed (Figs. 3 and 4). Greater differentiation among the
articular biochars being noted in the case of the test Microtox®
Fig. 3) than of the MARA test (Fig. 4). In the Microtox® test alliochars studied showed significant inhibition of luminescence of
BCW
Strains
1 2 3 4 5 6 7 8 9 10 11
Gro
wth
rela
tive
to c
ontro
l row
[%]
0
20
40
60
80
100
120
140
160
Gro
wth
rela
tive
to c
ontro
l row
[%]
BCM
Strains
1 2 3 4 5 6 7 8 9 10 11
Gro
wth
rela
tive
to c
ontro
l row
[%]
0
20
40
60
80
100
120
140
160
Gro
wth
rela
tive
to c
ontro
l row
[%]
Fig. 4. Effect of biochars’ leachates on microorganisms in MARA bioass
s Materials 260 (2013) 375– 382 379
V. fischeri. The strongest negative effect, as in relation to L. sativum,was observed for BC-M. In the case of that biochar, luminescenceinhibition was at the level of 99%. Only a slightly lower value (85%)was characteristic of biochar BC-2. For the remaining two biochars,i.e. BC-W and BC-O, inhibition of luminescence of V. fischeri was notas strong (Fig. 3) and was at the level of 40% and 12%, respectively.
In the MARA test, an evidently negative impact with relation toall the species was observed only for BC-O (Fig. 4). Biochars BC-Mand BC-2 also showed a toxic character towards more than a half ofthe test organisms. In the remaining cases, the values did not differsignificantly in relation to the control, or had a slight stimulatingeffect on the growth of the test microorganisms (BC-M – C. stos-teroni, BC-2 – Microbacterium sp.). Biochar BC-W was the only oneto display a distinctly positive impact and no differentiation amongthe test species (Fig. 4). The sensitivity of the particular test orga-nisms to the extracts depended also on the kind of material andwas closely related with the kind of biochar applied. The MARAassay generates a Toxic Unit (TU) for each of 11 microbial speciesexposed to biochar’s extract. The TU for the particular biochars wereas follows: BC-W < BC-2 < BC-M < BC-O. In accordance with the prin-ciple of the MARA assay, biochars with similar fingerprints maydisplay similarities in their toxic effects. The dendrogram resultingfrom cluster analysis of values determined for each biochar is ableto discriminate their (dis)similarities based on toxic fingerprinting(Fig. S1, electronic annex). BC-M, BC-O and BC-2 where closely clus-tered, which may suggest similar toxic effects, as opposed to BC-Wwhich indicates that it toxicity manifests differently to other testedbiochars.
Fig. 5A and B presents the toxicity of extracts from biochars with
mophila (Prototoxkit FTM). In both cases the lowest toxicity wascharacteristic of biochar BC-M, no negative effect of that biocharon the test organisms being found. The effect of the remaining
BCO
Stra ins
1 2 3 4 5 6 7 8 9 10 110
20
40
60
80
100
120
140
160
BC2
Stra ins
1 2 3 4 5 6 7 8 9 10 110
20
40
60
80
100
120
140
160
ay. Values are given as averages with standard deviations (n = 3).
380 P. Oleszczuk et al. / Journal of Hazardous Materials 260 (2013) 375– 382
BiocharsBC-W BC-O BC-M BC-2
Gro
wth
inhi
bito
n [%
]
0
5
10
15
20
25
30
35A
BiocharsBC-W BC-O BC-M BC-2
Mor
talit
y [%
]
0
5
10
15
20
25B
a
a
a
b
bb
cc
BiocharsBC-W BC-O BC-M BC-2
Mor
talit
y [%
]
0
20
40
60
80
100C
a
b
c
d
) and d
mi(mo2m(wta
caewdoo(wb
4
btetsacaacoaoMftftttnt
Fig. 5. Toxicity of raw (A – AlgaltoxkitFTM, B – ProtoxkitFTM
aterials clearly depended on the kind of assay. The highest toxic-ty towards S. capricornutum was characteristic of BC-O and BC-Wno significant differences between those biochars) which caused
ortality at the rates of 26.5% and 21.0%, respectively. The toxicityf BC-2 with relation to S. capricornutum was relatively low (at only%). As in the case of S. capricornutum, also with relation to T. ther-ophila the strongest toxicity was characteristic of biochar BC-O
mortality at the level of 16.8%). A similar rate of mortality, 15.1%,as noted with relation to BC-2. The toxicity of BC-W with relation
o T. thermophila was lower by more than a half compared to BC-Ond BC-2.
The most sensitive organism to the extracts from biochars wasrustaceans – D. magna. Raw extract obtained from BC-W, BC-Mnd BC-2 caused 100% mortality of those test organisms (Fig. S2,lectronic annex). Only in the case of BC-O relatively low valuesere observed, at the level of 10%. Double dilution of the extractid not produce any effect in the case of BC-M and BC-2, as the ratef mortality observed was still 100% (Fig. S2). Only 10-fold dilutionf the extract permitted a differentiation in the results obtainedFigs. 5C and S2). The results obtained revealed the same trend thatas observed in the case of V. fischerii. The toxicity of the particular
iochars was as follows BC-O < BC-W < BC-2 < BC-M.
. Discussion
Up to date no research has been conducted on the toxicity ofiochars. The available literature data relate primarily to the con-ent of polycyclic aromatic hydrocarbons [8,9,11,13] and, to a lesserxtent, trace metals [8]. The results obtained indicate that the con-ent of trace metals may differ significantly among the biocharstudied. However, both their level and thus the potential threat ofccumulation in soils during the application of biochars should notonstitute any real danger to living organisms (Table 2). Taking intoccount the level of biochars contamination with trace metals, theirddition to soil should not cause any significant increase in theirontent in the soil thus amended. The assayed values of the contentf trace metals were at a level similar to data presented by otheruthors [8]. Relatively higher levels of trace metals were noted forne of the biochars–that derived from miscanthus (BC-M) (Table 2).iscanthus is frequently used as an energy crop and grown in soils
ertilised with sewage sludge or wastewaters. Research shows thathe plant displays a high capability of accumulating trace metalsrom soils [30], which could have been related with its higher con-ent of those contaminants compared to the remaining materials
ested. It should be emphasised, however, that its content of metals,hough higher than in the other biochars under study, was still sig-ificantly lower than the permissible norms [31], and also lowerhan their average content in sewage sludges and composts [8]. The
iluted leachates (C – DaphtoxkitFTM) from tested biochars.
situation was totally different in the case of PAHs. Their content var-ied within a very broad range (Fig. 1), considerably exceeding theacceptable level [12] in the case of two biochars: BC-M and BC-2.The problem of biochar contamination by PAHs was deeply inves-tigated by Hale et al. [11]. Over 50 biochars were analysed and thetotal PAHs concentrations ranged from 70 to 3270 �g/kg. A similarlevel of the sum of PAHs (1480–5480 �g/kg) was also observed byCang et al. [32]. In a study conducted by Freddo et al. [8] it wasfound that the content of the sum of PAHs may vary, depending onthe kind of biochar, within the range from 80 to 8700 �g/kg. Dis-tinctly higher levels of the sum of PAHs in biochars were noted byHilber et al. [9] and Keiluweit et al. [10]. The values assayed by thoseauthors varied from 9110 to 355,300 �g/kg (sum of 16 PAHs), andfrom 30,500 to 50,000 �g/kg (sum of total PAHs and their meth-ylated derivatives). In the latter study the authors demonstrated adistinct relation between the content of PAHs and temperature ofpyrolysis. The highest levels of PAHs were noted in biochars pro-duced at temperature of 500 ◦C, i.e. lower than that applied in thisstudy. It should be emphasised that in a majority of the cases cited[8,10,11] biochars were obtained under controlled laboratory con-ditions. Only in the study by Hilber et al. [9] all biochars studiedby those authors originated from commercial suppliers, like thebiochars used in this study. This may suggest that biochars obtainedunder industrial conditions are characterised by PAHs levels higherthose in biochars obtained in a laboratory. This finds support in thestudy by Freddo et al. [8], where PAHs level in biochar obtained froma commercial manufacturer was distinctly higher than in biocharsproduced by the authors under controlled laboratory conditions.
The composition of the particular PAHs in the biochars understudy was also more similar to the results presented by Hilberet al. [9] and by Keiluweit et al. [10] than to the remaining stud-ies cited. Predominant in the biochars were 3-ring PAHs, primarilyphenanthrene. Whereas, on most of the biochars no presence ofnaphthalene was detected. Like in the study by Freddo et al. [8], nocontent of 6-ring PAHs was found. However, the PAHs compositionin biochars depended to a notable extent on the kind of materialfrom which the biochars were produced, which may explain theobserved differences with relation to the studies by other authors.This results support the hypothesis that the conditions under whichpyrolysis is conducted are also important.
At present there are no established norms concerning the per-missible levels of PAHs in biochars applied to soils. Solutionsproposed by the EU assume that the level of the sum of total PAHsin biochars should not exceed, depending on the proposal, from
6000 to 20,000 �g/kg [9]. At present biochars can be comparedto biosolids applied to soils, for which established norms alreadyexist. Maximum allowable concentrations for biosolids consideredfor the application to agricultural land are 6 mg/kg according to EU
P. Oleszczuk et al. / Journal of Hazardou
PAHs sum [ug /kg]
0 500 0 1000 0 1500 0 2000 0 2500 0 3000 0
D. m
agna
mor
talit
y [%
]
0
20
40
60
80
100
pb(gft
bmtTnomm(pwooac[wtt
ccboa
5
wdWib
[
[
[[
Fig. 6. The relationship between sum of 16 PAHs and mortality of D. magna.
roposal (for the sum of 11 PAHs) [12]. Only in the case of oneiochar its level of PAHs would disqualify it from agricultural useFig. 1). This is a very favourable result for the current situation ofrowing interest in the use of biochars. However, it indicates a needor research in this area, for the purpose of avoidance of potentialhreats.
Chemical analyses are common in the estimation of the applica-ility of various materials. However, only the use of biological testsakes it possible to better identify the potential risk involved in
he application of a given material for fertilisation purposes [17].he effect of the biochars studied on toxicity towards the test orga-isms was varied but, as can be assumed on the basis of the resultsbtained, closely related with the content of certain polycyclic aro-atic hydrocarbons (Fig. 6 and Table S2b – electronic annex). Theost sensitive organisms to the biochars proved to be D. magna
Fig. 6). Whereas, the biochars inhibited the growth of algae androtozoa at levels not exceeding 20% and 25%, respective (in theorst case). The literature does not provide any data on the toxicity
f biochars with relation to various organisms. Studies concerningther materials that can potentially play the role of fertilisers arelso limited. Relatively the most frequently applied are tests con-erning phytotoxicity [33] and tests with leachates from biosolids34]. The levels of toxicity obtained for the biochars under studyere similar to those observed for biosolids [17,35]. This indicates
hat biochars may have a significant effect on biological life also inhe negative sense.
For the purpose of estimation of the effect of the particularontaminants on the toxicity of the biochars studied analysis oforrelation was performed (Table S2). A significant correlationetween the toxic effect and the content of contaminants was notednly in the case of the sum of total PAHs (and selected compounds)nd the rate of mortality of D. magna (Fig. 6).
. Conclusion
To our knowledge, the results presented here are the first studyhere the ecotoxicological properties of different biochars were
etermined in the context of organic and inorganic pollutants.hile in the case of trace metals one should not expect any signif-
cant negative effect on the environment after the introduction ofiochars to soils, the content of polycyclic aromatic hydrocarbons
[
s Materials 260 (2013) 375– 382 381
may pose a real threat. Especially in the case of one biochar theassayed levels of PAHs notably exceeded the permissible limitsestablished for biosolids. Nevertheless, among the PAHs analysedno presence of 6-ring compounds was observed which are rel-atively stable and characterised by mutagenic and carcinogenicproperties. The level of PAHs in the biochars was relatively high,which may – after the application of the biochars to soils – sig-nificantly increase their content. This may constitute a threat toorganisms, which was confirmed by the ecotoxicological tests. Theliterature does not provide information on the subject of recom-mendations concerning the dosage of biochars.
Considering the results obtained it should be emphasised thatbiochars, like other fertilisers, should be subject to strict control andshould not be applied to soils without prior analyses. Of particularconcern is the negative effect of biochars on the ecotoxicologicalparameters tested. Although there are no official norms concerningthe application of biotests in the evaluation of biosolids, the resultsobtained indicate a risk of their negative effect on biological life.Biochars are usually applied in larger amounts than is the case withbiosolids. In this situation, norms concerning the content of PAHsand trace metals in biochars should take that problem into account.
Acknowledgments
This work was supported by a grant from Switzerland throughthe Swiss Contribution to the enlarged European Union.
Appendix A. Supplementary data
Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.jhazmat.2013.05.044.
References
[1] B. Glaser, J. Lehmann, W. Zech, Ameliorating physical and chemical propertiesof highly weathered soils in the tropics with charcoal – a review, Biol. Fertil.Soils 35 (2002) 219–230.
[2] K.Y. Chan, L. van Zwieten, I. Meszaros, A. Downie, S. Joseph, Agronomic values ofgreenwaste biochar as a soil amendment, Aust. J. Soil Res. 45 (2007) 629–634.
[3] J. Lehmann, S. Joseph, Biochar for Environmental Management, Earthscan Pub-lishers, London-Sterling, VA, 2009.
[4] L. Beesley, M. Marmiroli, The immobilisation and retention of soluble arsenic,cadmium and zinc by biochar, Environ. Pollut. 159 (2011) 474–480.
[5] P. Oleszczuk, S.E. Hale, G. Cornelissen, J. Lehmann, Activated carbon andbiochar amendments to decrease porewater concentration of polycyclic aro-matic hydrocarbons (PAHs) in sewage sludges, Bioresource Technol. 111 (2012)84–91.
[6] I. Hilber, T.D. Bucheli, Activated carbon amendment to remediate contaminatedsediments and soils: a review, Global NEST J. 12 (2010) 305–317.
[7] R.S. Kookana, The role of biochar in modifying the environmental fate, bioavail-ability, and efficacy of pesticides in soils: a review, Aust. J. Soil Res. 48 (2010)627–637.
[8] A. Freddo, C. Cai, B.J. Reid, Environmental contextualisation of potential toxicelements and polycyclic aromatic hydrocarbons in biochar, Environ. Pollut. 171(2012) 18–24.
[9] I. Hilber, F. Blum, J. Leifeld, H.P. Schmidt, T.D. Bucheli, Quantitative determina-tion of PAHs in biochar: a prerequisite to ensure its quality and safe application,J. Agric. Food Chem. 60 (2012) 3042–3050.
10] M. Keiluweit, M. Kleber, M.A. Sparrow, B.R.T. Simoneit, F.G. Prahl, Solvent-extractable polycyclic aromatic hydrocarbons in biochar: influence of pyrolysistemperature and feedstock, Environ. Sci. Technol. 46 (2012) 9333–9341.
11] S.E. Hale, J. Lehmann, D. Rutherford, A.R. Zimmerman, R.T. Bachmann, V. Shi-tumbanuma, A. O’Toole, K.L. Sundqvist, H.P.H. Arp, G. Cornelissen, Quantifyingthe total and bioavailable polycyclic aromatic hydrocarbons and dioxins inbiochars, Environ. Sci. Technol. 46 (2012) 2830–2838.
12] UE, European Union Draft Directive on Sewage Sludge, 2000, pp. 1–20, Brussels.13] S. Kloss, F. Zehetner, A. Fdellantonio, R. Hamid, F. Ottner, V. Liedtke, M. Schwan-
ninger, M.H. Gerzabek, G. Soja, Characterization of slow pyrolysis biochars:effects of feedstocks and pyrolysis temperature on biochar properties, J. Envi-
ron. Qual. 41 (2011) 990–1000.
14] E.B. Ledesma, N.D. Marsh, A.K. Sandrowitz, M.J. Wornat, Global kinetic rateparameters for the formation of polycyclic aromatic hydrocarbons from thepyrolysis of catechol, a model compound representative of solid fuel moieties,Energy Fuel. 16 (2002) 1331–1336.
15] O. Masek, M. Konno, S. Hosokai, N. Sonoyama, K. Norinaga, J.I. Hayashi, A studyon pyrolytic gasification of coffee grounds and implications to allothermal gasi-fication, Biomass Bioenergy 32 (2008) 78–89.
16] L. Koppolu, F.A. Agblevor, L.D. Clements, Pyrolysis as a technique for separatingheavy metals from hyperaccumulators. Part II. Lab scale pyrolysis of synthetichyperaccumulator biomass, Biomass Bioenergy 25 (2003) 651–663.
17] A. Malara, P. Oleszczuk, Application of a battery of biotests for thedetermination of leachate toxicity to bacteria and invertebratesfrom sewage sludge-amended soil, Environ. Sci. Pollut. Res. (2013),http://dx.doi.org/10.1007/s11356-11012-11268-11353 (in press).
18] L.P. van Reeuwijk, Procedures for Soil Analysis, ISRIC, Wageningen, 1995.19] S.J. Gregg, K.S.W. Sing, Adsorption, Surface Area and Porosity, 2nd ed., Academic
Press, London, 1982.20] Phytotoxkit, Seed germination and early growth microbiotest with higher
plants, in: Standard Operation Procedure, MicroBioTests Inc., Nazareth,Belgium, 2004, pp. 1–24.
21] EC, Council Decision of 19 December 2002 establishing criteria and proceduresfor the acceptance of waste at landfills pursuant to Article 16 of and Annex IIto Directive 1999/31/EC, 2003/33/EC, OJ L11, 2002, 27.
22] SDI, Microtox Manual, Microbics Corporation, Carlsbad, CA, 1992.23] K. Wadhia, T. Dando, K. Clive-Thompson, Intra-laboratory evaluation of Micro-
bial Assay for Risk Assessment (MARA) for potential application in theimplementation of the Water Framework Directive (WFD), J. Environ. Monit. 9(2007) 953–958.
24] Algaltoxkit, Freshwater toxicity tests with microalgae, in: Standard OperationalProcedure, Creasel, Deinze, Belgium, 1996, pp. 28.
25] OECD, Organisation for Economic Co-operation and Development, Guidelinesfor the Testing of Chemicals. Guideline 201. Alga, Growth Inhibition Test, 1984,14.
[
s Materials 260 (2013) 375– 382
26] F. Protoxkit, Freshwater toxicity test with a ciliaterprotozoan. Standard Oper-ation Procedure, Creasel, Deinze, Belgium, 1998.
27] F. Daphtoxkit, Crustacean Toxicity Screening Test for Fresh-Water. StandardOperational Procedure, Creasel, Deinze, Belgium, 1996.
28] OECD, Guidelines for the Testing of Chemicals. Guideline 202. Daphnia sp. AcuteImmobilisation Test and Reproduction Test, 1984.
29] B. Chen, D. Zhou, L. Zhu, Transitional adsorption and partition of nonpolarand polar aromatic contaminants by biochars of pine needles with differentpyrolytic temperatures, Environ. Sci. Technol. 42 (2008) 5137–5143.
30] P. Galbally, C. Fagan, D. Ryan, J. Finnan, J. Grant, K. McDonnell, Biosolidsand distillery effluent amendment to Irish Miscanthus ×giganteus planta-tions: impacts on groundwater and soil, J. Environ. Qual. 41 (2012) 114–123.
31] C.C.o.t.E. Communities, Directive of 12 June 1986 on the Protection of the Envi-ronment and in Particular of the Soil, When Sewage Sludge is Used in Soil, 1986,Brussels.
32] L. Cang, X. Zhu, Y. Wang, Z. Xie, D. Zhou, Pollutant contents in biochar andtheir potential environmental risks for field application, Nongye GongchengXuebao/Trans. Chin. Soc. Agric. Eng. 28 (2012) 163–167.
33] W.A. Ramirez, X. Domene, P. Andrés, J.M. Alcaniz, Phytotoxic effects of sewagesludge extracts on the germination of three plant species, Ecotoxicology 17(2008) 834–844.
34] H. Dizer, B. Fischer, I. Sepulveda, E. Loffredo, N. Senesi, F. Santana, P.-D. Hansen,Estrogenic effect of leachates and soil extracts from lysimeters spiked with
sewage sludge and reference endocrine disrupters, Environ. Toxicol. 17 (2002)105–112.
35] W.A. Ramirez, X. Domene, O. Ortiz, J.M. Alcaniz, Toxic effects of digested,composted and thermally-dried sewage sludge on three plants, BioresourceTechnol. 99 (2008) 7168–7175.
